Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA

Adenosine-to-inosine tRNA-editing enzyme has been identified for more than two decades, but the study on its DNA editing activity is rather scarce. We show that amphioxus (Branchiostoma japonicum) ADAT2 (BjADAT2) contains the active site ‘HxE-PCxxC’ and the key residues for target-base-binding, and amphioxus ADAT3 (BjADAT3) harbors both the N-terminal positively charged region and the C-terminal pseudo-catalytic domain important for recognition of substrates. The sequencing of BjADAT2-transformed Escherichia coli genome suggests that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome. Biochemical analyses further demonstrate that BjADAT2, in complex with BjADAT3, can perform A-to-I editing of tRNA and convert C-to-U and A-to-I deamination of DNA. We also show that BjADAT2 preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. Finally, we find that C89, N113, C148 and Y156 play critical roles in the DNA editing activity of BjADAT2. Collectively, our study indicates that BjADAT2/3 is the sole naturally occurring deaminase with both tRNA and DNA editing capacity identified so far in Metazoa.

Adenosine deaminases that act on tRNA (ADATs) perform a key function by deaminating the tRNA anticodon loop and allowing for wobble base pairing. While in bacteria this is the function of a single enzyme (TadA), in eukaryotes this functionality is performed by a heterodimer of two proteins that have cytidine deaminase folds. This prompted Alfonzo and colleagues, years ago, to look for cytidine deaminase functionality in these enzymes as well, which they found in DNA (but not tRNA). In this manuscript, Zhang and colleagues describe an ADAR2/3 homolog from amphioxus, that can catalyze A to I editing in tRNA. The ADAT2 portion of this heterodimer however, can perform C-to-U editing in DNA (similarly to the ADAT2 described in T.brucei) but also, uniquely, A-to-I editing in DNA. ADAT3 enhances, but is not required for activity. I have two comments: 1) experimentally, evidence of induction of mutation by deamination in the Ecoli but also yeast systems is usually supported by repeating the experiment in the context of a repair knockout that cannot deal with the specific mutation (UNG in the case of C to U, or AAG/MPG in the case of A to I). For completion, it would be important for the authors to do this.
2) The finding is important both for evolutionary biology but also for synthetic biology (as enzymes that can perform both functions have not been shown to exist in vivo; an evolved ADAR enzyme that can catalyze C to U editing has been reported by F. Zhang and colleagues, and it will be interesting for these authors to discuss them comparatively. Reviewer #2 (Remarks to the Author): In this manuscript, Zhan Gao et aｌ. investigated amphioxus Adenosine-to-inosine tRNA-editing enzyme 2 (BjADAT2) and showed that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome and preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. This manuscript is a well-done enzymological study and examines the characteristics of BjADART2 in detail. However, this manuscript merely examines an enzyme in a previously unexamined animal species for ADAT2 that has already been identified, lacks impact. The authors studied DNA editing in eukaryotic cells of BjADAT2, but does it edit its own genomic DNA? If there is a possibility, the heterogeneity of the genomic DNA of the amphioxus should be proven. If there is no such possibility, then the meaning of the study in natural science is unclear. If the research is to utilize BjADAT2 as a genome editing tool, it is necessary to show its superiority compared to other tools (enzymes), but such research has not been done. Therefore, I recommend that the authors submit it to a more suitable journal. Comments 1. The authors show the 3D structures of BjADAT2 in Fig. 1, how were these results obtained? Also, is there any reason to believe that these structures, especially the surface view on the right, is correct? 2. In Fig. 2, the authors show mutation frequencies and distributions, but how many clones were analyzed for each study? 3. In line 161 and 162, the authors suggested that BjADAT2 might prefer targeting the 5'-TC context while deaminating cytidine in DNAs. The results certainly suggest this, but how does it work for double-stranded DNAs, where the bases are inside of the sugar backbone and should form complementary base pairs with each other?
Gao reported "Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination". DNA editing activity triggered by adenosine-to-inosine tRNA-editing enzyme has been harnessed as genome editing tool, while deaminase from nature possessing high DNA editing have not been identified. In the present study, authors tested BjADAT2 and BjADAT3, resulting in the identification of BjADAT2 possessing relative high DNA editing. In general, the study is well designed and well performed. While, there are several issues which should be addressed. 1. A positive control(i.e,.ecTadA from ABE8e) for the A to G editing activity should be included in at least one study, thus, we could assess the relative editing activity of BjADAT2. 2. In Fig 1b, it is better to add human ADAT2 alignment. 3. As to the study of BjADAT2 in E. coli and yeast, is there any recombination between endogenous ADAT2 and BjADAT2? Or BjADAT2/ADAT3 compound dimer? 4. tRNA editing activity of ADAT2 is as we expected, which may not be treated as a selling point in this study. Thus, this section could be minimized. 5. The biological function of BjADAT2/ADAT3 may be further discussed although so far it is not clear.

Reviewer #4 (Remarks to the Author):
Reviewer's comments: The manuscript has been written so nicely and I really congratulate the authors for bringing a very nice topic which will contribute a lot to the scientific society. Although some minor revisions are needed for the upgrading the quality of the manuscript. Recently some works have been published regarding the C to U and A to I editing which has really made a huge contribution and I think those citations are needed and at the same time at the discussion part some relevant sentences needed to be added. Such as: https://doi.org/10.3390/genes13091636 RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code | Scientific Reports (nature.com) https://doi.org/10.1093/protein/gzz005 Moreover, the result section could be in a more detailed form and in more relevance with the discussion. Finally, I would like to say that with those minor revisions this manuscript can be accepted in this reputed journal. I would like to revise the manuscript after the revision.

Reviewer #1:
Adenosine deaminases that act on tRNA (ADATs) perform a key function by deaminating the tRNA anticodon loop and allowing for wobble base pairing. While in bacteria this is the function of a single enzyme (TadA), in eukaryotes this functionality is performed by a heterodimer of two proteins that have cytidine deaminase folds. This prompted Alfonzo and colleagues, years ago, to look for cytidine deaminase functionality in these enzymes as well, which they found in DNA (but not tRNA). In this manuscript, Zhang and colleagues describe an ADAR2/3 homolog from amphioxus, that can catalyze A to I editing in tRNA. The ADAT2 portion of this heterodimer however, can perform C-to-U editing in DNA (similarly to the ADAT2 described in T.brucei) but also, uniquely, A-to-I editing in DNA. ADAT3 enhances, but is not required for activity. I have two comments: 1) experimentally, evidence of induction of mutation by deamination in the E. coli but also yeast systems is usually supported by repeating the experiment in the context of a repair knockout that cannot deal with the specific mutation (UNG in the case of C to U, or AAG/MPG in the case of A to I). For completion, it would be important for the authors to do this.
Response: Thanks for the suggestion. We have performed the analysis of rpoB mutation for Rif R in an E. coli strain lacking alkAgene (alkA gene encodes an alkyadenine DNA glycosylase, i.e., AAG), and the result provided further evidence on the adenosine deaminase activity of BjADAT2 (the primary highlight of this manuscript). Please see the revised manuscript. For the experiment of cytidine deamination, our study here (including the mutational spectrum and distribution in the rpoB and gyrA genes) has demonstrated the cytidine deaminase activity of BjADAT2. In addition, to unbiasedly determine the deaminase activity, we analyzed the genome-wide mutations using a Cirseq method, and the results provided strong evidence that BjADAT2 could deaminate cytidine and adenosine in the E. coli genome. Therefore we did not re-do the experiment in the ungbackground.
2) The finding is important both for evolutionary biology but also for synthetic biology (as enzymes that can perform both functions have not been shown to exist in vivo; an evolved ADAR enzyme that can catalyze C to U editing has been reported by F. Zhang and colleagues, and it will be interesting for these authors to discuss them comparatively.
Response: Thanks. We have discussed the deaminases with both A to I and C to U editing activities comparatively. Please see the section of the revised manuscript.

Reviewer #2:
In this manuscript, Zhan Gao et al. investigated amphioxus Adenosine-to-inosine tRNA-editing enzyme 2 (BjADAT2) and showed that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome and preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. This manuscript is a well-done enzymological study and examines the characteristics of BjADART2 in detail. However, this manuscript merely examines an enzyme in a previously unexamined animal species for ADAT2 that has already been identified, lacks impact. The authors studied DNA editing in eukaryotic cells of BjADAT2, but does it edit its own genomic DNA? If there is a possibility, the heterogeneity of the genomic DNA of the amphioxus should be proven. If there is no such possibility, then the meaning of the study in natural science is unclear. If the research is to utilize BjADAT2 as a genome editing tool, it is necessary to show its superiority compared to other tools (enzymes), but such research has not been done. Therefore, I recommend that the authors submit it to a more suitable journal.
Response: Thanks for the comments and suggestions. We have performed the experiments as suggested. We have analyzed the amphioxus somatic mutations and discussed the possible biology functions of BjADAT2/3. Please see the last paragraph of the Discussion section. In brief, somatic single-base substitution frequency in amphioxus (10 -3 ) is orders of magnitude higher than that of other multicellular eukaryotes (10 -5~1 0 -7 ). Among the single-base substitutions, C:G to T:A and A:T to G:C are dominant types with the frequency of 8.3×10 -3 and 6.6×10 -3 , respectively ( Supplementary Fig. S9), implying a high frequency of cytidine and adenosine deamination on amphioxus genomic DNA. We are currently investigating the mutational mechanism in amphioxus and its relevance with BjADAT2/3 in future.
Comments 1. The authors show the 3D structures of BjADAT2 in Fig. 1, how were these results obtained? Also, is there any reason to believe that these structures, especially the surface view on the right, is correct?
Response: The 3D structure model of BjADAT2 was predicted using I-TASSER program (Yang and Zhang, Nucleic Acids Research, 2015, 43:W174-181), followed by quality assessments using QMEANDisCo and ProQ3D servers, which confirmed that it was good in quality. The model here is to show that BjADAT2 contains a core fivestranded β sheet structural element surrounded by α helices, as well as the active site 'HxE-PCxxC' within the potential substrate-binding pocket formed by the loop 1, loop 3, loop 5, loop 7, and C-terminal helix. The information can be completely embodied by the cartoon view in Fig. 1a. Given that the surface view on the right is just another form of the cartoon view, we have removed the surface view, and it has no effects on the conclusions of molecular characteristics and any other analyses.
2. In Fig. 2, the authors show mutation frequencies and distributions, but how many clones were analyzed for each study?
Response: Thanks. The description of the number of clones has been added in the figure legends. The mutagenic activity assay in E. coli (Fig. 2a-c) was performed according to the classical method (Petersen-Mahrt et al., Nature, 2002, 418:99-103). In Fig. 2a, each group contains eight independent cultures (each point represents the mutation frequency of an independent culture). In Fig. 2b and 2c, the data are collected from 50 independent cultures of BjADAT2-transformed E. coli and an equal number of controls (one clone per culture). Similarly, 30 independent cultures are collected for each group in Fig. 2d. In Fig. 2e-g, the data of each group are collected from three independent cultures.
3. In line 161 and 162, the authors suggested that BjADAT2 might prefer targeting the 5'-TC context while deaminating cytidine in DNAs. The results certainly suggest this, but how does it work for double-stranded DNAs, where the bases are inside of the sugar backbone and should form complementary base pairs with each other?

Response:
We have modified this sentence. Our study demonstrated that the targeting of BjADAT2 is single-stranded DNAs. Previous studies also reported that many deaminases, e.g., AID and APOBECs, target single-stranded DNAs (Chaudhuri et al., Nature, 2003, 422:726-730;Salter et al., Trends in Biochemical Sciences, 2016, 41:578-594). We speculate that BjADAT2 can deaminate cytidine and adenosine when single-stranded DNAs are exposed, such as in the process of DNA transcription or replication. We appreciate your valuable comments, and will continue to study the mutagenic mechanism and physiological function of BjADAT2 in future.

Reviewer #3:
Gao reported "Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination". DNA editing activity triggered by adenosineto-inosine tRNA-editing enzyme has been harnessed as genome editing tool, while deaminase from nature possessing high DNA editing have not been identified. In the present study, authors tested BjADAT2 and BjADAT3, resulting in the identification of BjADAT2 possessing relative high DNA editing. In general, the study is well designed and well performed. While, there are several issues which should be addressed.
The manuscript has been written so nicely and I really congratulate the authors for bringing a very nice topic which will contribute a lot to the scientific society. Although some minor revisions are needed for the upgrading the quality of the manuscript. Recently some works have been published regarding the C to U and A to I editing which has really made a huge contribution and I think those citations are needed and at the same time at the discussion part some relevant sentences needed to be added. Such as: https://doi.org/10.3390/genes13091636 RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code | Scientific Reports (nature.com) https://doi.org/10.1093/protein/gzz005 Moreover, the result section could be in a more detailed form and in more relevance with the discussion. Finally, I would like to say that with those minor revisions this manuscript can be accepted in this reputed journal. I would like to revise the manuscript after the revision.
Response: Thanks for the comments. We have modified the section Discussion and cited these wonderful works.